METHODS OF INVESTIGATION. 15 



III. METHODS OF INVESTIGATION. 



In the study of any problem dealing with the development of fluid-spaces within 

 the body, the method of investigation must of necessity be such as to offer excep- 

 tional opportunities for control. In the present work several well-known and 

 generally accepted anatomical procedures were naturally suggested, such as injec- 

 tion of the spaces about the central nervous system, reconstruction from serial 

 sections, or merely study of the various stages by means of serial sections. 



It was ascertained early in the investigation that by injection and serial sec- 

 tions without reconstruction the necessary stages in the process of meningeal differ- 

 entiation could be established. In regard actually to the physiological aspects of 

 the problem more reliance was placed on the results of injection than on any histo- 

 logical differentiation, for, as explained above, considerations of the pathway and 

 of the flow of the cerebro-spmal fluid were deemed most important. No method 

 of injection, however, holds out much promise in such a problem unless it can be 

 applied, under conditions approximating the normal, within the spaces about the 

 nervous system. The greatest objection to reliance upon injections in this problem 

 is in relation to pressures. From the very nature of the case it will be realized that 

 any ordinary injection into the embryonic central canal or perispinal space must 

 result in an extraordinary increase in the normal tension of the fluid. This objec- 

 tion applies to any method employed, whether that of a simple syringe and needle, 

 the glass tube and bulb devised by Knower^, or a glass capillary-tube contrivance. 



The erroneous conclusions drawn by investigators from the employment of 

 excessive pressures of injection are nowhere more strikingly illustrated than in 

 studies of the circulation of the cerebro-spinal fluid. Many such examples were 

 recently brought forward in a critical review^ 55 ^ published in connection with a study 

 of the fluid. In the embryo, with structures and membranes still of very little 

 tensile strength, the consequences of a disregard for the pressures of injection are 

 even more disastrous. 



A second criterion for the study of fluid-pathways in the body is necessarily 

 the type of injection mass. Not only should attention be paid to the pressures 

 involved, but the peculiarities of the particular body-fluid concerned must be con- 

 sidered. Adopting for this work on the embryo the same standards followed in the 

 previous investigation on the adult, true solutions were used in place of the customary 

 granular suspensions. Emulsions and viscous solutions were not employed because 

 of their obvious disadvantages in studying the passage through membranes. India 

 ink and process black (in which carbon granules are the particulate matter) were also 

 used, but only for comparison with the standard true solution, as the likelihood 

 of the insoluble granules being phagocyted within the period of experimentation 

 or of being caught mechanically in tissue meshes appeared a priori to be too great. 



In any study of fluid-pathways in the body, not only must the injection fluid 

 be a true solution, but it must also be one which is not attracted to particular cells 

 (as with many stains) . Likewise, colloid stains (such as the benzidene group) could 

 not be employed, because of the fact that certain cells (macrophages, as described 



